There are a number of bead-based assay technologies used to study biomolecular interactions in a microplate format, for example, AlphaScreen® and AlphaLISA®, manufactured by PerkinElmer of Waltham, Mass. The acronym “Alpha” stands for amplified luminescent proximity homogeneous assay. These technologies are non-radioactive, homogeneous proximity assays. Binding of molecules captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a detectable luminescent/fluorescent signal, which provides qualitative and quantitative information about one or more analytes in a sample.
AlphaScreen® and AlphaLISA® assays each utilize two bead types: Donor beads and Acceptor beads. Donor beads comprise a photosensitizer, for example, phthalocyanine, which converts ambient oxygen to an excited and reactive form of oxygen, singlet oxygen, upon illumination at 680 nm. Singlet oxygen is not a radical; it is molecular oxygen with a single excited electron. Like other excited molecules, singlet oxygen has a limited lifetime prior to falling back to ground state. Within its 4 μsec half-life, singlet oxygen can diffuse approximately 200 nm in solution, as compared to TR-FRET which has a maximum transfer distance of about 10 nm. If an Acceptor bead is within that proximity, energy is transferred from the singlet oxygen to thioxene derivatives within the Acceptor bead, subsequently culminating in light production within a range of wavelengths, e.g., 520-620 nm (AlphaScreen®) or at a particular wavelength, e.g., 615 nm (AlphaLISA®). In the absence of an Acceptor bead, singlet oxygen falls to ground state and no signal is produced. This proximity-dependent chemical energy transfer is the basis for AlphaScreen's homogeneous nature, such that no washing steps are required, unlike ELISA assays, electrochemiluminescence, and flow cytometry assays, thereby offering a significant advantage.
Both AlphaScreen® and AlphaLISA® rely on the same Donor beads yet use different Acceptor beads. AlphaScreen® Acceptor beads are embedded with three dyes: thioxene, anthracene, and rubrene. Rubrene, the final fluor, emits light detectable between 520-620 nm. In the AlphaLISA® Acceptor beads, anthracene, and rubrene are substituted with an Europium chelate. The Europium (Eu) chelate is directly excited by the 340 nm light resulting from the conversion of thioxene to a di-ketone derivative following its reaction with singlet oxygen. The excited Europium chelate generates an intense light detectable within a much narrower wavelength bandwidth centered around 615 nm. In contrast to the AlphaScreen®, the AlphaLISA® emission is therefore less susceptible to interference by either artificial or natural compounds (such as hemoglobin) that absorb light between 500-600 nm.
AlphaScreen® and AlphaLISA® are typically run as multi-well (e.g., 96-, 384-, or 1536-well) assays, and are used to perform both biochemical and cell-based assays. They can be used for low to high affinity binding interactions (e.g., pM to mM), and can be used for high-throughput screening (HTS). AlphaLISA® is compatible with complex matrices such as cell lysates, serum, plasma, CSF, and the like. These systems can perform immunoassays, epigenetic assays, kinase assays, antibody detection and characterization, immunogenicity, selective detection of sAPP and amyloid peptides, alpha protease assays, alpha ligand-receptor assays, cAMP assays, cGMP assays, and detection of protein-protein and protein-nucleic acid interactions.
AlphaPlex™, manufactured by PerkinElmer of Waltham, Mass., is a homogeneous multiplexing reagent technology that utilizes the above-described alpha technology. By using multiple Acceptor beads which emit different wavelengths, multiple analytes can be detected. The system offers accurate multiplex quantification of a wide range of analytes, from large proteins to small proteins and scarce biological samples such as primary cells and stem cells, and is applicable to a wide range of applications including biomarkers for PD/PK, biomarkers for stem cells, kinase (e.g., total vs. phosphorylated protein), epigenetic markers (e.g., total histone vs. specific marker), amyloid peptides, IgG profiling, and assay normalization with housekeeping proteins.
The existing systems that utilize alpha technology are not portable. Crop sciences and animal health researchers/technicians are required to procure samples in the field, then take them back to the laboratory for analysis. Many samples may need to be taken to insure that a particular analyte of interest will be present in at least some of the samples when they are taken back to the lab for analysis.
Portable aminoassay devices include lateral flow devices, e.g., an aminoassay run in a cartridge, such as a pregnancy test, where the sample reacts with an antibody and produces a visible color when the analyte is present in the sample. Other portable systems involve dipsticks, e.g., paper or plastic embedded with reagents, which are dipped into a solution for determination of the presence of an analyte in the sample. These systems are typically not very accurate, and are usually limited to qualitative analyses.
There is a need for robust, easy-to-use, portable assay systems and devices that are more accurate and more sensitive than existing portable systems.